Labeled Immunoassays Study Notes

Chapter Overview

• Labeled immunoassays overview.

• Types of immunoassays:

  • Heterogeneous vs. homogeneous assays.

  • Competitive vs. noncompetitive immunoassays.

  • Various specific types:

  • Radioimmunoassay.

  • Enzyme immunoassays (EIAs).

  • Interferences in EIAs.

  • Chemiluminescent assays.

  • Fluorescent immunoassays.

  • Rapid immunoassays.

Labeled Immunoassays

• Purpose and design:

  • Designed for detecting antigens and antibodies, typically small in size or present at low concentrations.

  • Utilize a labeled reactant (detection molecule) to assess specific binding amounts.

  • Common analytes detected include microbial antigens, hormones, drugs, tumor markers, and specific immunoglobulins.

Immunoassay Labels

• Different types of labeling substances used:

  • Enzymes: Enzyme/colorimetric substrate.

  • Chemiluminescent molecules: Chemiluminescent molecule combined with a trigger solution.

  • Fluorophores: Fluorescent compounds.

  • Radioactive isotopes: Older methods typically involving radioimmunoassay.

Heterogeneous vs. Homogeneous Immunoassays

• Heterogeneous immunoassays:

  • Require physical separation of bound and free components.

  • Usually involve binding to solid phases such as polystyrene reaction wells, microparticle beads, latex beads, or plastic tubes.

  • Techniques may include magnetic separation or centrifugation.

• Homogeneous immunoassays:

  • Do not require physical separation.

Competitive Immunoassays

• Characteristics:

  • All reactants mixed simultaneously.

  • Labeled and unlabeled antigens compete for a limited number of binding sites on reagent antibodies.

  • The amount of bound label is inversely proportional to the concentration of the labeled antigen.

  • These assays have high specificity and are ideal for measuring small, pure antigens (e.g., drugs and hormones).

Competitive Immunoassay Principle

• Process description:

  1. Unknown concentration of analyte in a patient sample competes with a labeled analyte for binding sites on an immobilized antibody.

  2. Wash step to remove unbound materials.

  3. Substrate is added, generating a colored product (signal) whose intensity correlates to the amount of enzyme-labeled analyte bound to the antibody.

  4. Signal strength is inversely related to analyte concentration.

  • Diagram referenced shows comparative analyte concentration:

  • Sample A (Negative Control) has low concentration.

  • Sample B (Patient Sample) has higher concentration.

Noncompetitive Immunoassays

• Also known as capture, sandwich, or immunometric assays:

  • Patient antigens are bound by antibodies attached to a solid phase.

  • After a wash step to eliminate unbound antigens, a labeled second antibody is introduced.

  • The amount of label correlates directly with the amount of antigen in the patient sample.

Noncompetitive Immunoassay Principle

• Process description:

  1. Reagent antigen immobilized on solid phase binds antibodies from the patient sample.

  2. Wash to remove unbound materials (step may be optional for one-step assays).

  3. Introduce an enzyme-labeled (detection) antibody that binds to the human immunoglobulin.

  4. Another wash to clear unbound materials.

  5. Add substrate to measure signal (e.g., color intensity), which is directly proportional to the concentration of antibody in the patient sample.

Radioimmunoassay (RIA)

• Historical significance:

  • First immunoassay developed.

  • Utilizes radioactive labels, primarily ^{125}I.

• Mechanism:

  • Emits gamma radiation detectable by a gamma counter.

  • Highly sensitive and precise; ideal for measuring trace amounts of analytes (e.g., hormones, serum proteins, drugs).

• Competitive mechanism:

  • Amount of label in the bound phase indirectly proportional to patient antigen concentration.

• Disadvantages:

  • Health hazards associated with radioactive substances.

  • Disposal issues regarding low-level radioactive waste.

  • Limited shelf life of some reagents.

Enzyme Immunoassays (EIAs)

• Sensitivity and design:

  • Highly sensitive assays using enzyme labels that react with substrates to produce detectable breakdown products that are chromogenic, fluorogenic, or luminescent.

  • Available in both competitive and noncompetitive formats.

• Commonly used enzymes:

  • Alkaline phosphatase.

  • Horseradish peroxidase.

  • Glucose-6-phosphate dehydrogenase (G6PDH).

  • β-D-galactosidase.

Indirect ELISA

• Mechanism:

  • Noncompetitive immunoassay used for detecting antibodies in patient samples, such as for hepatitis B or autoantibodies (e.g., antinuclear antibodies, thyroglobulin antibody).

Indirect ELISA Principle

• Commonly automated due to:

  • High sensitivity and specificity.

  • Simplicity of use.

  • Cost-effectiveness.

Capture (Sandwich) Immunoassays

• Purpose:

  • Used to detect antigens in patient samples, especially for antigens with multiple determinants (e.g., cytokines, proteins, tumor markers).

  • Can also be used for specific immunoglobulins (e.g., IgM for acute infections).

• Process:

  • Sample antigen binds to an antibody attached to the solid phase.

  • After incubation, an enzyme-labeled antibody is added, completing the “sandwich.”

  • Substrate addition produces a colored or chemiluminescent product detectable as a measure of enzyme activity directly proportional to antigen amount.

Biotin-Avidin Labeling

• Components involved:

  • Biotin: Vitamin B7 (also known as Vitamin H).

  • Streptavidin (SAv): A bacterial protein that binds with high affinity to biotin.

• Application:

  • Biotin can be attached to antibodies and streptavidin to solid-phase materials to enhance signaling and sensitivity in ELISAs and capture immunoassays.

Interferences With Immunoassays

• Types of interference:

  • Caused by properties of the specimen.

  • Antigen interference.

  • Antibody interference.

• Example:

  • High doses of biotin supplements can interfere with assays employing biotin-SAv labeling, potentially leading to false results (either positive or negative).

High-Dose Hook Effect

• A phenomenon:

  • Excess patient antigen can cause falsely low detection, where analyte concentrations seem low or normal when they are actually high.

Antibody Interferences

• Common issues:

  • Autoantibodies like rheumatoid factor can induce false positives.

  • Heterophile antibodies typically yield false-positive results (e.g., human and mouse antibodies - HAMA).

Homogeneous EIAs

• Features:

  • Generally less sensitive than heterogeneous assays.

  • Rapid and simple to perform.

  • Include EMIT and CEDIA classifications.

• Utility:

  • Primarily used for determining low-molecular-weight analytes in serum and urine, including hormones, therapeutic drugs, and drugs of abuse.

  • No washing or separation steps are necessary.

• Mechanism:

  • Enzyme activity correlates directly with patient antigen or hapten concentration in test solutions, with antibody binding blocking the active site on the enzyme, resulting in measurable activity loss.

General Principle of a Homogeneous Immunoassay

• Process description:

  • Reagent antibody is in solution; patient antigen and enzyme-labeled antigen are added to the test tube.

  • Both compete for a limited set of antibody binding sites.

  • If patient antigen is present, the enzyme label on the reagent antigen remains unblocked, resulting in color development.

  • Color depth indicates the concentration levels: Sample A (low concentration) shows less color, whereas Sample B (high concentration) displays stronger color.

Chemiluminescent Immunoassays

• Overview:

  • Highly sensitive automated immunoassays detecting antigens (like therapeutic drugs and steroid hormones) or antibodies.

• Mechanism:

  • Involves light emission through a chemical reaction, typically an oxidation reaction, with an excited molecule that reverts to the ground state.

• Common chemiluminescent molecules:

  • Acridinium esters.

  • Ruthenium derivatives.

  • Nitrophenol oxalates.

• Application types:

  • Applicable to both heterogeneous and homogeneous assays.

Chemiluminescent Microparticle Immunoassay (CMIA)

• Description:

  • A heterogeneous assay where patient antigen competes with chemiluminescent-labeled antigen for antibody-coated microparticles.

  • Magnetic separation utilized for cleaning and washing.

Electrochemiluminescence Immunoassay (ECLIA)

• Mechanism:

  • Ruthenium labels take part in a chemical reaction at the surface of an electrode.

Fluorescent Immunoassays

• Functionality:

  • Employ fluorochromes as the label.

  • Fluorochromes absorb energy from incident light and emit it at a longer wavelength (lower energy) when electrons return to the ground state.

• Example compounds:

  • Fluorescein: Absorbs light at 490–495 nm, emits green light at 520 nm.

  • Rhodamine: Absorbs light at 550 nm, emits red light at 585 nm.

Direct Immunofluorescence Assays

• Usage:

  • Identifying pathogens in patient samples.

  • Fluorescent tag-conjugated antibodies are added to fixed tissue sections or cells on a microscope slide.

• Process:

  • After incubation, a wash step is executed, and reading occurs using a fluorescence microscope.

• Application:

  • Fluorescent-labeled antibodies for CD antigens to identify lymphocytes and other cells via flow cytometry.

Indirect Immunofluorescence Assays

• Procedure:

  • Patient serum incubates with a microscope slide that has a known antigen attached.

  • After washing, an anti-human immunoglobulin with a fluorescent tag is added, forming a sandwich with the initial antibody.

• Technique use cases:

  • Identify patient antibodies (e.g., ANAs) through this methodology.

Direct vs. Indirect Immunofluorescence Assays

• Comparison highlights:

  • Direct Assay: Patient antigen directly interacts with fluorescent-labeled antibody on a fixed slide, followed by washes. Fluorescence indicates the presence of antigens.

  • Indirect Assay: Specific patient antibodies bind to antigens on fixed slides, followed by addition of labeled anti-human immunoglobulin. A wash step follows to remove excess labeling; the resulting fluorescence is proportional to the patient antibody level.

• Note:

  • Subjectivity in slide reading is a challenge across immunofluorescence assays.

Multiplex Immunoassay (MIA)

• Definition:

  • Fluorescent immunoassay enabling simultaneous detection of multiple antibodies or antigens.

• Mechanism:

  • Patient serum incubates with polystyrene beads conjugated to various antigens.

  • Addition of a fluorescent-tagged anti-human immunoglobulin reveals antibody binding, with beads distinguished by shade in flow cytometry.

Rapid Immunoassays

• Characteristics:

  • Membrane-based, single-use assays employing immunochromatography.

  • Easy execution with a quick turnaround time; suited for point-of-care testing.

• Procedure:

  • Patient sample added to test membrane, combining with labeled antigen or antibody linked to colored latex or colloidal gold particles.

  • Immune complexes form and migrate across membrane, producing a colorimetric reaction.

Rapid Immunoassays: Immunochromatography

• Process steps depicted:

  • (A) Patient sample is introduced into a cassette with antibody labeled by colloidal gold.

  • (B) Sample interacts with antibody and is propelled by capillary flow.

  • (C) Monoclonal antibody attached to the analyte captures patient antigen associated with gold-labeled antibody.

  • (D) Control line contains antibody that captures the colloidal gold-labeled antibody, indicating assay validity.